| Electroacupuncture(EA)is an effective method for pain therapy.Some researchers used classical physical approaches and pharmacological approaches to identify that EA analgesia(EAA)is involved in some cerebral nuclei or areas.However,the function of a single nucleus or area cannot elucidate the neural mechanism underlying EAA.EAA-related neural circuitries thus need to be further studied.Increasing studies demonstrated that EA could induce releasing of various analgesic substances(e.g.enkephalin,endorphin)and anti-analgesic substances(e.g.cholecystokinin-octopeptide-8,orphanin FQ and antiotensin ?).Obviously,EAA should be a comprehensive manifestation mediated by multiple substances in CNS.Since neurons in different nuclei or areas vary in function and possess complex intracellular molecules,central molecular mechanism by which EA mediates is largely unknown.The aim of present study was to investigate the involvement of neural circuits,central substances and signal pathways in EAA.1.EA activated common neural circuitsTo investigate the common neural circuits in EAA,the present study performed the neurons labeling technique to explore the differentially activated nucleus and areas in goats.Twenty-eight crossbred male goats(30 ± 2 kg)were randomly assigned for 4 groups: the blank control,the sham control,EA at Baihui and Santai acupoints and EA at bilateral Housanli acupoints.The goats in EA group were treated with EA one time(60Hz,30min).Goats in the control group were restrained as those treated in EA group.Goats in the sham group were treated with needles left on acupoints but without electricity.The pain thresholds were measured with the potassium iontophoresis method,which was recorded before and after EA.The change in pain threshold was calculated and was presented as the EA analgesic effect.After pain thresholds test,all goats were immediately euthanized and their brain and spinal samples were collected.The levels of c-Fos in the caudate nucleus,the nucleus accumbens,the lateral septal nucleus,the medial septal nucleus,the paraventricular nucleus of the hypothalamus,the ventromedial nucleus of the hypothalamus,the arcuate nucleus,the nucleus amygdala basalis,the lateral habenula nucleus,the parabrachial nucleus,the locus coeruleus,the nucleus tractus solitarius,the anterior lobe of the pituitary gland,the caudal ventrolateral periaqueductal grey(vl PAG),the nucleus raphe magnus(NRM),the gigantocellular reticular nucleus(GI)and spinal cord dorsal horn(SCDH)were determined with Streptavidin-Biotin Complex immunohistochemistry.The results showed that the pain threshold in the two EA groups increased when compared with the blank control.Pain threshold induced by EA at set of Baihui-Santai acupoints was higher than that by EA at set of Housanli acupoints.Compared with blank control,EA at two sets of acupoints increased c-Fos expression in the medial septal nucleus,the arcuate nucleus,the nucleus amygdala basalis,the lateral habenula nucleus,the locus coeruleus,the pituitary gland,vl PAG,NRM,and SCDH.Compared with EA at set of Housanli points,EA at set of Baihui-Santai points induced more c-Fos expression in the nucleus amygdala basalis,but less in the nucleus amygdala basalis,the paraventricular nucleus of the hypothalamus,the lateral habenula nucleus and SDH.It suggests that the arcuate nucleus-periaqueductal grey-nucleus raphe magnus / locus coeruleus-spinal cord dorsal horn and the hypothalamus-pituitary may be the common activated neural pathways taking part in EA-induced analgesia at the two sets of acupoints.2.Electroacupuncture-induced gene differential expression in the periaqueductal grey of goatsTo explore the full profile of EA-induced molecular modification in the central nerve system,the present study performed the deep sequencing technique to explore the differentially expressed genes(DEGs)in goats.These DEGs were validated by q PCR.Among them,the role of thymosin beta 4 in EAA was further confirmed through intracerebroventricular(icv)injection technique.In the experiment of RNA-sequencing,three pairs of goat twins were selected for the match-paired experiment.One of the paired goats from the same litter was treated with EA and another was used as control.The rats in EA groups were treated with EA one time(60Hz,30min).The pain thresholds were measured with the potassium iontophoresis method,which was recorded before and at 0 h and 4 h after EA,and the change in pain threshold was calculated.The goats were sacrificed at 4 h after the experiment,and the periaqueductal gray(PAG)were harvested for RNA-sequencing.DGEs were analyzed using GO and KEGG functional annotations.Fifteen genes were selected and validated with q PCR.Among these genes,thymosin beta 4 was selected and its roles in EAA were confirmed through icv injection technique in the physiological and pathological model.In the physiological model,72 SD rats(250 ± 20 g)were divided into two groups(n=36/group): EA group and control group.The rats in EA or the control group received icv 1 ?g/?L T?4,0.1 ?g/?L T?4,0.01 ?g/?L T?4,Lip-T?4-si RNA,Lip-C-si RNA(negative si RNA)or PBS,with each reagent injected to 6 rats.Then the rats in EA group received one time of EA stimulation.The tail flick latency(TFL)was recorded before and after EA,and the change in TFL was calculated.In the pathological model,54 SD rats(250 ± 20 g)were divided into three groups: CFA+EA group(n=36 rats),CFA+CON group(n=36 rats)and Saline+CON group(n=6 rats).The rats in CFA+EA or the CFA+CON groups were treated with 0.1m L CFA paw injection,and received icv 0.1 ?g/?L T?4,Lip-T?4-si RNA,Lip-C-si RNA or PBS injection,with each reagent injected to 6 rats.The rats in Saline+CON group were treated with 0.1m L saline paw injection,and received icv PBS injection.Then the rats in CFA+EA group received EA at 0 d,2 d,4 d,6 d,8 d,10 d and13 d.The paw withdrawal threshold(PWT)was recorded before and at 1,3,7,14 d after CFA or saline injection.The change in PWT was calculated.Results demonstrated that pain threshold of goats in EA group increased(p < 0.01)pain threshold compared with the control goats.Total 2,651 differentially expressed genes(DEGs),including 1,709 up-regulated and 852 down-regulated genes,were found and enriched in 30 KEGG pathways and 149 gene ontology(GO)terms.EA-induced up-regulated(met-enkephalin,proopiomelanocortin,preprodynorphin,opioid receptor kappa 1,glutamate receptor 1,excitatory amino acid transporter 1,gamma-aminobutyric acid type B receptor subunit 1,Glial fibrillary acidic protein,Aromatic-L-amino-acid decarboxylase and Synaptotagmin-1)and down-regulated(Acyl-Co A-binding protein,proprotein convertase 1 inhibitor,Thymosin beta-4,Thymosin beta-10 and prostaglandin F synthase)genes were validated with realtime PCR.The results were consistent with their expression changes as identified with RNA-sequencing.Furthermore,results exhibited that up-regulated glutamate receptors and transporters,GABA receptors and transporters,synaptotagmins and MAPKs might contribute to EAA through the functional categories related to Glutamatergic synapse,GABAergic synapse,mitogen-activated protein kinases,ribosome and ubiquitin-proteasome pathways.The results of icv injection experiment in physiological model showed that,TFL of rats in EA+T?4 group was lower than TFL of rats in EA+PBS group(p < 0.05).In inflammatory model,PWT of rats in CFA+EA+T?4 group was lower than PWT of rats in CFA+EA+PBS group at 1,3 and 14 d after CFA injection(p < 0.05).PWT of rats in CFA+EA+Lip-T?4-si RNA group was lower than PWT of rats in CFA+EA+PBS group at 1,3,7 and 14 d after CFA injection(p < 0.05).These results suggested the involvement of T?4 in EAA under physiological and inflammatory condition. |
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